1. Field of the Invention
The present invention relates generally to the fields of biochemistry and cell biology. More particularly, it concerns novel cross-linking reagents for use in identifying molecular interactions and linking known molecules.
2. Description of Related Art
Large, multi-protein complexes mediate most important biological processes. A central question in the study of these xe2x80x9cprotein machinesxe2x80x9d (Alberts, 1998) is to elucidate the lacework of protein-protein interactions and, ideally, to ascertain how this architecture might change during the course of the catalytic cycle in question. Chemical cross-linking (Mattson et al., 1993; Ji and Ji, 1989; Gaffnley, 1985) is a potentially useful technique for this purpose. However, cross-linkers are most often applied to the analysis of interactions between a few proteins (Bertrand et al., 1997; Jiang et al., 1993; Jiang and Kodadek, 1993), while successful analysis of contacts in large complexes is more difficult (Friedrichson and Kurzchalia, 1998; Schere and Krieg, 1991; Norcum and Warrington, 1998). This is largely due to the fact typical bifunctional cross-linkers, comprised of two electrophiles connected by a linker arm, have many drawbacks with regard to complex systems. For example,they are constitutively reactive and cannot be xe2x80x9ctriggeredxe2x80x9d at a desired time. Also, poor yields are often obtained even with long incubation times. Even more seriously, large-scale modification of nucleophilic side chains, such as the acylation of lysines, on the surface of the proteins during the extended incubation times required for typical reagents raises the concern of artifactual results due to structural destabilization.
Protein-protein interactions vary in their strength of association by several orders of magnitude. These associations can be so strong as to be functionally irreversible or weak enough as to allow only transient association between proteins. Weak interactions between proteins can often times play fundamental roles in biologic systems. However, understanding these interactions and dissecting the relevant proteins involved in the interactions is problematic. Trapping such proteins involved in weak interactions with chemical cross-linkers is a powerful way of identifying them.
Another instance where weak protein interactions are encountered is in obtaining probes or reagents that bind target proteins. Methodologies for generating peptides that bind a target are known. Antibody generation against a target has been one of the preferred methods for generating such high binding affinity probes, several novel approaches have become routine. These include systems for screening large numbers of peptides for binding including recombinant antibody generation, yeast two-hybrid systems, phage display and combinatorial libraries. Screens for binding can either be genetic or involve high throughput assays to isolate peptides or polypeptides with binding specificities for a target. One problem often seen with peptides or polypeptides is weak affinity for the target against which they are directed. Cross-linking reagents can be used to trap these weak interactions, turning a probe with modest affinity and limited utility into a more useful reagent.
A final scenario where crosslinkers can be used is in combination with known compounds, peptides or proteins to covalently trap these compounds in key molecular interactions. In this scenario, a known or candidate compound would be conjugated with a crosslinking agent prior to adding to a biologic system. Upon addition and binding to a target such as a receptor, enzyme, or any other target site, crosslinking would permanently attach the compound, peptide or protein to its target. This would be especially useful in vivo for increasing efficacy of pharinaceutical compositions.
There are many cross-linkers that have been characterized for defined purposes. Use of these cross-linkers is limited by many factors, including non-specific reactivity, constitutive reactivity, harsh chemical conditions necessary for reaction, destructive modifications of target enzymatic activity, and an inability to use the cross-linkers in complex biologic systems such as in whole cells or in vivo. Thus, there is a need for cross-linkers that do not suffer from these drawbacks.
The present invention describes methods of covalently bonding a first molecule to a second molecule in a controlled way. The method comprises bringing the two molecules in molecular proximity to each other, contacting the two molecules with a metal-ligand complex, and subjecting the complex to light which photoactivates the complex, resulting in bonding the first and second molecules mediated by the metal-ligand complex. In preferred embodiments, the first molecule is a protein, an antibody, drugs, or a peptide. The peptide may be conjugated to a metal. In other preferred embodiments, the second molecule is a protein.
The present invention describes complexes useful for bonding two molecules together that comprising a palladium(II) porphyrin or a Ru(II)(bypyridyl) used in combination with an electron acceptor. In preferred embodiments, the electron acceptor is ammonium persulfate. The complex may also comprises a detectable agent.
The light used to photoactivate the complex has a wavelength of greater than 380 nm but less than 800 nm. The intensity of the light used to photoactivate the complex is between 1 watt and 1000 watts for periods of time between 30 milliseconds to 30 seconds.
The present application also describes methods where the first and second molecules are located in a cell. The complex-linked molecules may be isolated allowing identification of the first and/or second molecule.
In other embodiments of the present invention, an alternative method of covalently bonding a first molecule to a second molecule is described. This involves initially forming a metal-ligand complex with a first molecule, then contacting this first molecule with a second molecule, subjecting the complex to light to photoactivate the complex, resulting in bonding of the first molecule to the second molecule. In preferred embodiments, the first molecule is a protein, an antibody, or a peptide. The peptide may be conjugated to a metal. In other preferred embodiments, the second molecule is a protein. In other embodiments, the second molecule is a drug.